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Biomedical subjects

A Meister

Publications and source records attributed to A Meister.

At least 37 records · Page 2Linked to original sources
PubMed1999

The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.

Amino Acid Sequence↗
PubMed1999

Antispasmodic activity of Thymus vulgaris extract on the isolated guinea-pig trachea: discrimination between drug and ethanol effects.

The effect of an extract of Thymus vulgaris on induced spasms was investigated on guinea-pig trachea preparations. By the experimental setup used, effects of ethanol as the vehicle could be differentiated from the activity of the herbal constituents. The extract reversibly and concentration-dependently antagonized the contraction of the Musculus transversus tracheae, provoked by four different spasmogens (BaCl2, carbachol, histamine, prostaglandin F2 alpha). The degree of the antispasmodic activity was dependent on the individual spasmogen with prostaglandin F2 alpha being most efficiently antagonized.

Animals↗
PubMed1998

Prevention of lithium nephrotoxicity in a novel one-hour model in rats.

It is well established that lithium can cause morphologically visible damage to the kidneys of humans and animals. Although the clinical significance of its nephrotoxicity is debatable, it would be desirable to find a method to prevent lithium's effect on the kidneys. Toward this end, we have developed a novel method for producing nephrotoxicity that will be useful for research on prevention. A single, large, toxic dose of lithium chloride (LiCl) caused necrosis of the distal convoluted tubules, which was visible by light microscopy in 30 min, had fully developed in 1 h, and had disappeared by the next day. The lesions were seen after i.p. or i.v. injections of fasted rats of three different strains. Equivalent doses of NaCl, KCl, MgCl2 and combinations thereof had no such effect, nor did they inhibit nephrotoxicity when incorporated into the LiCl solution. However, relatively small doses of LiCl injected by any route 3 or 24 h beforehand prevented the nephrotoxicity. The mechanism of prevention is not known, but it does not involve reduction of lithium levels in the kidneys.

Animals↗
PubMed1998

[Radiotherapy for choroidal neovascularization in age-related macular degeneration. A pilot study using low- versus high-dose photon bean radiation].

PURPOSE: Several pilot studies have indicated that low-dose radiation therapy might have a beneficial effect on the course of choroidal neovascularization (CNV) in age-related macular degeneration (AMD). This study aimed to ascertain whether such treatment might halt the progression of neovascular AMD and whether a low or a high radiation dose should be applied. PATIENTS: The patients comprised some randomized to 0 vs 10 vs 36 Gy of radiation and (after a change of the study protocol became necessary) others who participated in a prospective, controlled non-randomized pilot study. Enclosed were eyes with visual acuity of > or = 0.1 and < or = 0.6 revealing a juxta-subfoveal CNV either of the occult type (type 1) or the classic type (isolated or as part of a predominantly occult lesion). RESULTS: Eyes treated with 10 Gy for occult CNV (n = 12) were subject to severe visual loss in 41.6% of the cases compared to 38.5% in the control group (n = 13) at 12 months of follow-up. For eyes treated with 10 Gy because of classic CNV, the corresponding figures were 33% (n = 18) and 57% (n = 14) respectively. At 18 months of follow-up, the percentages were 63% and 75% respectively. Fluorescein angiographic growth of classic and occult CNV could not be halted by 10 Gy, while a temporary growth retardation was observed in cases irradiated with 36 Gy. CONCLUSION: In the study presented, the natural course of occult CNV could not be improved by irradiation with 10 or 36 Gy. In cases of classic CNV, low-dose irradiation with 10 Gy postponed severe visual loss by a maximum of 18 months. A positive treatment effect was also observed in cases irradiated with 36 Gy; however, a 25% incidence of radiation retinopathy seems unacceptable.

Aged↗
PubMed1997

Influence of body size on lithium levels in rats.

Lithium chloride was injected into rats by the intraperitoneal or intravenous route. The dose was proportional to body weight, in the conventional manner. Lithium levels in blood serum and organs were determined after 3-24 h. Within a given strain, large rats had higher levels than small rats. The size of the rats, and not their age, was the determining factor. The large rats had more adipose tissue than the small rats. Inasmuch as lithium distributes in body water, the excess fat in large rats reduces its volume of distribution, which may be responsible for raising the lithium levels in aqueous compartments, including serum. Male and female rats of equal body size developed equal lithium levels in serum.

Age Factors↗
PubMed1997

Neural network method to determine the vigilance levels of the central nervous system, related to occupational chronic chemical stress.

The effects of chronic toxic occupational factors and functional disorders of the central nervous system (CNS) in chemical industry were studied. These factors cause various stages of chronic chemical stress on the human CNS together with changes of the vigilance levels. On the basis of QEEG data analysis and psychometric tests we identified three stages of occupational chemical stress syndromes according to the CNS vigilance level (ordered from light to severe): hypersthenic syndrome, hyposthenic syndrome, and organic psychosyndrome. Each syndrome is characterized by specific changes in the QEEG data. A perceptron-based neural network was developed for the classification of the QEEG data to one of the above-mentioned syndrome classes. The data of 77 patients and 10 healthy subjects were selected to test the algorithm. Different combinations of the QEEG data as input features to the classifier were chosen. The most reliable classification was obtained when QEEG data measured during the visual stimulation of the CNS were used. However, sometimes the algorithm was unable to solve the classification problem, or it took a very long time to train the perceptron. In part, difficulties arose from using a perceptron-based algorithm, which can classify only linearly separable data.

Algorithms↗
PubMed1996

The amino acid sequence of rat kidney 5-oxo-L-prolinase determined by cDNA cloning.

5-Oxoprolinase (EC 3.5.2) catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic hydrolysis of ATP to ADP and inorganic phosphate. Highly purified preparations of the enzyme have been obtained from rat kidney and Pseudomonas putida. The rat kidney enzyme is composed of two strongly interacting, apparently identical subunits (Mr = 142,000), whereas that from P. putida is composed of two functionally different protein components that can readily be dissociated. Here we report the cloning of rat kidney 5-oxoprolinase with preliminary expression studies. cDNA clones encoding the enzyme were isolated by screening a lambdagt11 cDNA library beginning with a degenerate oligonucleotide probe based on peptide sequence data obtained from the purified enzyme. The whole cDNA clone was completed by amplifying its 5' end from a premade library of rat kidney Marathon-ReadyTM cDNAs using polymerase chain reaction methodology. The composite cDNA (4,016 bases) revealed an uninterrupted open reading frame encoding 1,288 amino acid residues (Mr = 137,759). The deduced amino acid sequence contains all four of the peptide sequences that were independently found in peptide fragments derived from the enzyme. Expression of the full-length clone in Escherichia coli yielded a product of the same size as the rat kidney enzyme and which reacted with antibodies directed against the rat kidney enzyme. The predicted amino acid sequence is almost 50% identical throughout its entire length to that of a hypothetical yeast protein YKL215C. It is also 26% identical in half its length to the bacterial hydantoinase HyuA and 26% identical in the other half to the bacterial hydantoinase HyuB. The results suggest unexpected evolutionary relationships among the hydantoinases and rat kidney 5-oxoprolinase which share the common property of hydrolyzing the imide bond of 5-membered rings but which do not all require ATP.

Amino Acid Sequence↗
PubMed1996

Low mutagenic effects of mitomycin C in undifferentiated embryonic P19 cells are correlated with efficient cell cycle control.

Pluripotent undifferentiated embryonic carcinoma cells of line P19 and their differentiated progeny, epithelioid ectoderm-like EPI-7 cells, showed different responses to mitomycin C (MMC) with respect to induction of micronuclei, mutations at the HPRT-locus and cell cycle control. Cytotoxic effects of MMC after a 5-h treatment were lower in undifferentiated P19 cells than in differentiated EPI-7 cells with IC50 values of 1.3 and 0.25 microM for P19 and EPI-7 cells, respectively. MMC did not induce 6-thioguanine-resistant mutants in P19 cells but significantly increased the mutation frequency in EPI-7 cells with concentrations of 0.25, 0.5 and 1.0 microM MMC. Micronuclei determined by flow-cytometry were induced by MMC in both cell lines at equitoxic concentrations of 4.5 (P19) and 0.75 (EPI-7) microM, reducing the viability in both cell lines to 10%. Whereas the induction of micronuclei in P19 cells was maximal 28 h after treatment and declined thereafter, micronucleus induction peaked 48 h post treatment in EPI-7 cells and remained significantly increased even 67 h after the treatment. Flow-cytometric determination of the distribution of MMC-treated P19 and EPI-7 within the cell cycle revealed a distinct G2/M-block in P19 cells, whereas EPI-7 cells showed normal progression through S-phase and a negligible G2/M-block. Therefore, we conclude that the lower effectivity of MMC to induce gene mutations and micronuclei in P19 cells seemed to be correlated with a more efficient cell cycle control in undifferentiated compared to differentiated EPI-7 cells.

Animals↗
PubMed1996

Flow cytometry of mesophyll and bundle sheath chloroplast thylakoids of maize (Zea mays L.).

Chlorophyll fluorescence at short and long wavelengths was used to sort thylakoid membranes of maize, a plant with the C4 dicarboxylic acid pathway of photosynthesis, in a flow cytometer. The method yielded two distinct particle populations that were identified as mesophyll and bundle sheath thylakoids by low-temperature fluorescence spectroscopy and by the pigment ratio of chlorophyll a/b. Mesophyll and bundle sheath thylakoids were essentially pure after sorting by flow cytometry. Fluorescence data and chlorophyll a/b pigment ratios of thylakoids separated by flow cytometry were compared with earlier data of preparations obtained by conventional isolation procedures. Our results indicate that impure mesophyll and bundle sheath membranes were used in most previous investigations. We were unable to detect the major light-harvesting complex of PS II (LHC II) in our pure bundle sheath thylakoids using fluorescence excitation spectroscopy. Therefore, we believe that the previously reported presence of LHC II in bundle sheath chloroplasts of maize can be attributed to mesophyll contamination.

Chloroplasts↗
PubMed1996

Measurement of phospholipase A2 and 1-alkylglycerophosphocholine acetyltransferase activities in stimulated alveolar macrophages by HPLC analysis of NBD-labeled ether lipids.

The importance of phospholipases in cellular signaling and 1-alkylglycerophosphocholine acetyltransferase in the formation of platelet-activating factor (PAF) has stimulated demand for methods to measure these enzyme activities in inflammatory cells. Most of the assays currently used rely on radiolabeled substrates. We have synthesized NBD-labeled ether lipids as substrates for measuring enzyme activities of the PAF cycle and of lysosomal phospholipase A2 (PLA2). The fluorescent lipids were incubated with homogenates of stimulated bovine alveolar macrophages. The generated products were separated from the substrates by HPLC on a normal phase and monitored with a fluorescence detector. NBD-lyso-PAF was well accepted by acetyl- and acyltransferases of the cell-free preparations, which metabolized the substrate into NBD-PAF and NBD-alkyl-acylglycerophosphocholines. Homogenates of stimulated cells showed an enhanced production of NBD-PAF. The increased formation of the biological mediator was dependent on the nature of the stimuli and the time of stimulation. Lysosomal PLA2 was measured with 1-O-(12-NBD-aminododecyl)-2-acyl-sn-glycero-3-phosphocholine as substrate. By varying the pH and the calcium concentration, it was possible to distinguish between the cytosolic PLA2 and the lysosomal PLA2 activity. Optimal conditions for the determination of the lysosomal PLA2 were obtained at pH 4.5 and in the presence of EDTA. Stimulation with particulate agonists induced an enhancement of the lysosomal PLA2 activity in macrophages.

4-Chloro-7-nitrobenzofurazan↗
PubMed1996

Primordial germ cell-derived mouse embryonic germ (EG) cells in vitro resemble undifferentiated stem cells with respect to differentiation capacity and cell cycle distribution.

Embryonic germ (EG) cells of line EG-1 derived from mouse primordial germ cells were investigated for their in vitro differentiation capacity. By cultivation as embryo-like aggregates EG-1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue-specific genes and proteins as shown by RT-PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI-7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI-7 cells showed a longer G1-phase and shorter S- and G2/M-phases. Together, our results demonstrate that the differentiation state and capacity of EG cells in vitro resemble that of totipotent ES cells.

Animals↗
PubMed1996

Betaine generation in cardiac myocytes after adrenergic activation of phosphatidylcholine hydrolysis.

In this report, effects of alpha 1-adrenergic stimulation on phosphatidylcholine (PC) hydrolysis and the subsequent generation of water-soluble choline metabolites were investigated after preincubation of isolated cardiac myocytes of adult rats with [methyl-3H]choline. Choline uptake into cardiac myocytes was apparently mediated by a choline carrier which could be inhibited by hemicholinium-3. Analysis of the intracellular choline metabolites was performed by HPLC. Adrenergic stimulation of cardiac myocytes by (-)-phenylephrine, which is also known to activate the phosphoinositide signaling system, induced the generation of betaine as a selective signal transduction response. Agonist-induced generation of betaine in cardiac myocytes was maximal at 10 min after stimulation, and was optimal at physiologically relevant (-)-phenylephrine concentrations (1-10 microM). Betaine accumulation was transient, and no betaine remained detectable after 15 min. CDP-choline, however, was still elevated after 15 min which is indicative of continued PC resynthesis after adrenergic stimulation. The source of betaine in cellular signalling appeared to be hydrolysis of membrane PC to phosphatidic acid and choline by phospholipase D with subsequent oxidation of choline to betaine. This is based on the observation that radioactivity in unstimulated cells is present only in the lipid phase (presumably as PC) or as phosphocholine in the aqueous phase of the cells. The latter finding suggests that choline is rapidly phosphorylated after uptake into cardiac myocytes. Collectively, these results suggest a hypothetical role of betaine in the cellular signal transduction response to alpha 1-adrenergic stimulation in cardiac myocytes.

Adrenergic alpha-Agonists↗
PubMed1996

Lithium distribution in experimental inflammation of brain and spinal cord.

1. Autoimmune inflammation of the nervous system caused extensive changes in the distribution of lithium injected into rats. 2. Serum lithium levels were greatly increased because of failure of renal excretion caused by pre-renal azotemia, urinary retention and lack of dietary sodium. Brain, spinal cord, pituitary and adrenal levels of lithium were also elevated, reflecting the high serum levels. 3. However, the location and degree of this elevation corresponded to the predominant location of the inflammation. As a result, lithium levels in spinal cord approached and even exceeded the lithium content of brain.

Animals↗
PubMed1996

The flow karyotype of Arabidopsis thaliana interphase chromosomes.

The DNA contents of various aneuploid lines of Arabidopsis thaliana were measured by flow cytometry of 4',6-diamidino-2-phenylindole-stained interphase nuclei in suspensions and compared with each other as well as with the wild-type. The fluorescence intensifies for all lines were highly reproducible as were the deviations from the wild-type. The results allowed the estimation of the relative DNA contents of each Arabidopsis chromosome and of chromosomes arms. The sum of the surplus values for all trisomics was close to the value expected for the haploid (2C) DNA content. Only the line with the smallest telotrisome (Tr 3A) did not significantly differ in DNA content from that of the wild-type. It is concluded that approximately 3% of the genome represents the limit for resolution of differences in DNA content in this system. Thus, the approach allows a fast and reliable screening for duplications and deficiencies extending to 3% of the Arabidopsis genome. Regarding chromosomes sizes a comparison of the flow karyotype with existing karyotypes revealed differences which are discussed.

Aneuploidy↗
PubMed1995

Involvement of Ser-451 and Ser-452 in the catalysis of human gamma-glutamyl transpeptidase.

The serine residue required for catalysis of gamma-glutamyl transpeptidase was identified by site-specific mutagenesis of the conserved serine residues on the basis of sequence alignment of the light subunit of human, rat, pig and two bacterial enzymes. Recombinant human gamma-glutamyl transpeptidases with replacements of these serine residues by Ala were expressed using a baculovirus-insect cell system. Substitutions of Ala at Ser-385, -413 or -425 yielded almost fully active enzymes. However, substitutions of Ala at Ser-451 or -452 yielded enzymes that were only about 1% as active as the wild-type enzyme. Further, their double mutant is only 0.002% as active as the wild type. Kinetic analysis of transpeptidation using glycylglycine as acceptor indicates that the Vmax values of Ser-451 and -452 mutants are substantially decreased (to about 3% of the wild type); however, their Km values for L-gamma-glutamyl-p-nitroanilide as donor were only increased about 5 fold compared to that of the wild type. The double mutation of Ser-451 and -452 further decreased the Vmax value to only about 0.005% of the wild type, while this mutation produced only a minor effect (2-fold increase) on the Km value for the donor. The kinetic values for the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide in the mutants followed similar trends to those for transpeptidation. The rates of inactivation of Ser-451, -452 and their double mutant enzymes by acivicin, a potent inhibitor, were less than 1% that of the wild-type enzyme. The Ki value of the double mutant for L-serine as a competitive inhibitor of the gamma-glutamyl group is only 9 fold increased over that of the wild type, whereas the Ki for the serine-borate complex, which acts as an inhibitory transition-state analog, was more than 1,000 times higher than for the wild-type enzyme. These results suggest that both Ser-451 and -452 are located at the position able to interact with the gamma-glutamyl group and participate in catalysis, probably as nucleophiles or through stabilization of the transition state.

Amino Acid Sequence↗
PubMed1995

Human gamma-glutamyl transpeptidase mutants involving conserved aspartate residues and the unique cysteine residue of the light subunit.

Mutant human gamma-glutamyl transpeptidases with amino acid substitutions on the light subunit at the Asp residues conserved among several species, and at the unique cysteine residue (Cys-454), were prepared and expressed in a baculovirus insect cell system. Replacement of Asp-423 by Ala or Glu led to major loss of enzyme activity, consistent with the conclusion that Asp-423 is essential for activity. A mutant in which Cys-454 was replaced by Ala was fully active, indicating that the unique light subunit thiol is not required for catalysis. Kinetic analysis of the hydrolysis reaction of L-gamma-glutamyl-p-nitroanilide indicated that the decreased activity of Asp-423 mutants is the consequence of an extremely high substrate Km value, which is more than a 1000-fold greater than that for the wild-type enzyme, whereas the Vmax is decreased only less than 90-fold. The results suggest that Asp-423, and to a lesser extent Asp-422, interact electrostatically with the alpha-amino group of the gamma-glutamyl donor substrate. Although further studies are required to evaluate the possibility that the reaction involves function of a charge (or proton) relay system, the present work suggests that the gamma-glutamyl moiety of the substrate binds electrostatically to specific groups on the enzyme; this facilitates gamma-glutamyl enzyme formation.

Aspartic Acid↗